Iron binding agents

ABSTRACT

Composition, article of manufacture for and method of treating malaria in a human having an infestation of  Plasmodium  protozoans are described. The method comprises administering a therapeutically-effective amount of a compound of formula (I) or (IV), i.e. sufficient quantity to reduce the population of  Plasmodium . The composition of the invention is a compound of formula (I) or (IV) with a pharmaceutical excipient. The article of manufacture is the composition in combination with labeling for treating malaria. The substituents are detailed in the specification.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/993,417, filed Nov. 19, 2004 now U.S. Pat. No. 7,144,904, whichis a continuation application of U.S. application Ser. No. 10/216,492,filed Aug. 8, 2002 now U.S. Pat. No. 6,864,270 which is a continuationof U.S. application Ser. No. 09/723,809, filed Nov. 28, 2000, nowabandoned, which is a continuation of PCT/US99/21726, filed Sep. 21,1999, which claims the benefit of U.S. Provisional Application Ser. No.60/101,321, filed Sep. 21, 1998, the entire teachings of which areincorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by Grant Nos.3203522-12, ROIHL42817 and ROIDK49108 from the National Institutes ofHealth (NIH). The Government has certain rights in the invention.

TECHNICAL FIELD

This invention relates to the treatment of malaria with pharmaceuticalcompositions comprising certain compounds related to desferrithiocin.The compositions are particularly useful for the treatment of Plasmodiumfalciparum malaria.

BACKGROUND OF THE INVENTION

Malaria is one of the oldest and most widespread infectious diseasesplaguing mankind. Among human parasitic diseases, it is the most deadly.It is endemic in developing countries and infects over 500 millionpeople each year, killing over 2.7 million of them. Thanks in part tonew treatments for the disease, during the middle part of the 20^(th)century the incidence of malaria was decreasing each year. In recentyears, however, cases of malaria have dramatically increased worldwideincluding thousands of cases in the United States. This increase is duein part to the emergence of drug resistant strains of the disease.

Malaria is caused by eukaryotic protozoans of the genus Plasmodium. Ofthe 100 species of Plasmodium, four are known to cause malaria inhumans. Three of these species, Plasmodium vivax, Plasmodium malariae,and Plasmodium ovale cause relatively benign forms of the disease. Thefourth species, Plasmodium falciparum, is malignant and the most lethal,being responsible for the majority of deaths from malaria world wide.

P. falciparum malaria is introduced into human hosts through a bite fromthe female Anopheles mosquito. An infected mosquito will bite a humanand inject a small amount of saliva with anticoagulant and haploidsporozoites of the P. falciparum parasite. The sporozoites enter thecirculatory system and reach the liver in an hour or so. In the liverthey will enter hepatic parenchymal cells in what is called theexoerythrocytic stage of the disease cycle. During the 5-7 days of thisstage, they will undergo multiple asexual fission, schizogony,multiplying 30,000 to 40,000-fold, and produce merozoites.

As merozoites, they will leave the liver, reenter the bloodstream,invade erythrocytes (red blood cells), and begin the erythrocytic stage.Once inside the erythrocyte, P. falciparum begins to enlarge as anuninucleate trophozoite. Over another 1-3 days, this trophozoite willdivide asexually to produce a schizont containing 6-24 nuclei. Theschizont will divide and produce mononucleated merozoites. This causesthe erythrocyte to lyse and release merozoites into the blood stream toinfect other erythrocytes.

Some merozoites will differentiate into macrogametocytes andmicrogametocytes, which do not cause erythrocytes to lyse. These maleand female sexual forms can be ingested by a mosquito, in which theywill be fertilized to form zygotes which will produce sporozoites in themosquito's salivary glands to permit reinfection of other human hosts.

Malaria is asymptomatic until the erythrocytic stage when thesynchronized release of merozoites and debris from erythrocytes into thecirculation causes the classical malarial signs and symptoms. Theseinclude paroxysms (spasms and convulsions), high fever, rigors(stiffness and chills), profuse sweating, vomiting, anemia, headache,muscle pains, spleen enlargement, and hypoglycemia. Since the release ofmerozoites occurs every 48 hours or so in P. falciparum malaria, thesymptoms are tertian, occurring every third day. In between merozoitereleases of the erythrocytic stage, a human host will feel normal and beasymptomatic.

The most severe consequence of P. falciparum malaria is the aggregation,clumping, or sludging of infected erythrocytes, including adherence toblood vessel walls. Depending on the site of the sludging, lifethreatening effects can occur due to the restriction of blood flow tovital organs. These include encephalopathy for cerebral malaria,pulmonary edema, acute renal failure, severe intravascular hemolysis,and hemoglobinuria. The vast majority of deaths caused by P. falciparummalaria are due to these effects.

Traditional treatments of malaria are based on either the control ofmosquito populations, vaccines, or chemotherapy. For chemotherapy, drugsare generally targeted at specific stages of the disease. Such drugsinclude tissue schizonticides, such as chloroquine, used to eradicatethe exoerythrocytic stage in the liver; blood schizonticides, such aschloroquine, folate antagonists, and the 8-aminoquinolines referred toas pyrimethamine, primaquine, and pamaquine, used to destroy theerythrocytic stage; gametocytocides, such as 4-aminoquinolines, used tokill gametocytes; and sporonticides used to kill sporozoites.

In recent years, the most effective treatment for malaria, particularlyfor P. falciparum malaria, has been the 4-aminoquinoline, chloroquine.This drug of choice to treat the disease is active against theerythrocytic form of P. vivax and P. falciparum. Chloroquine acts as ablood schizonticidal agent and rarely produces serious side effects. Itinhibits nucleic acid and protein synthesis in protozoal cells. It isused both for the treatment of acute onset malignant tertian P.falciparum malaria and prophylactically.

It has been the prophylactic use of many chemotherapeutic treatments formalaria that has led to the emergence of drug resistant strains ofPlasmodium species that cause malaria. Plasmodium resistance tochloroquine has now become widespread and is a serious problem. This haslead to the development of alternative chemotherapeutic agents.

Compounds to emerge include folate antagonists, including sulfones andsulfonamides, such as dapsone, sulfadoxine, sulfadiazine, and sulfalene;primines, and biguamides. These compounds compete with p-aminobenzoicacid (PABA), interfere with synthesis of tetrahydrofolic acid, and actas blood schizonticides. However, their effective doses can be extremelytoxic and Plasmodium can readily develop resistance to these drugs.

The ability of Plasmodium species to develop resistance to drugs coupledwith the undesirable side effects of such drugs has resulted in theconstant development of new treatments. Thus, there are numerouscompounds currently available, or in development, for the treatment ofmalaria.

Antiprotozoal compounds that can be or have been used as treatmentsagainst malaria may be found by referring to Goodman and Gilman's ThePharmacological Basis of Therapeutics, Eighth Edition, McGraw-Hill, Inc.(1993), Chapter 41, pages 978-998.

As indicated above, strains of P. falciparum that are resistant to oneor more of the available treatments for malaria are ubiquitous today. Asnew compounds to attack the parasite directly are developed, a newresistant strain emerges. Additionally, the continued undesirable sideeffects of available drugs present problems. This is particularly truewhen multiple drugs must be administered to battle concurrent infectionsof more than one Plasmodium species, which have become quite common.Thus, not only are yet more alternative chemotherapeutic treatments formalaria desired, particularly for P. falciparum malaria, but alsoentirely new mechanisms of action for the eradication of the Plasmodiumparasite are desired. Such mechanisms may make it more difficult forstrains of the parasite to emerge that are resistant to these new drugs.

OBJECTS OF THE INVENTION

One object of this invention is to provide a new family of antimalarialswhich are particularly active against P. falciparum and yet haverelatively low toxicity over the treatment regimen.

Another object of this invention is to provide a new method for treatingmalaria using the new family of antimalarials.

Another object of this invention is to provide a novel method for thetreatment of P. falciparum malaria, particularly in strains of P.falciparum that are resistant to traditional chemotherapeuticantimalarial agents.

Other objects will become apparent to one of ordinary skill in the artupon reading the following disclosure.

SUMMARY OF THE INVENTION

One aspect of the invention is an antimalarial composition comprising acompound, represented by formula (I), in combination with apharmaceutically acceptable excipient. The formula is

In the formula (I), the substituents are defined as follows:

R is OH, OR₇, or N(OH)R₈;

R₁ is H, CH₃ or an available electron;

R₂ is. H, CH₃ or an available electron;

R₃ is H, CH₃ (as the (R) or (S) configuration) or an available electronand together with either R₁ or R₂ when one is an available electron,forms a double bond with the R₁/R₂ carbon;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 14 carbons, O-alkyl of 1-4 carbons, or(CH₂)_(a)(R₁₀)_(b)(CH₂)_(a)R₁₀(CH₂)_(a)(R₁₀)_(b)X;

R₆ is H, OH, alkyl of 1-6 carbons, a halogen, (CH₂)_(a)R₁₀(CH₂)_(r)R₁₀Y,or is —C═C—C═C—, which, together with R₁₁ when R₁₁ is an availableelectron, forms a fused ring system as follows:

R₇ is alkyl of one to four carbons or optionally substituted benzyl;

R₈ is H, alkyl of one to four carbons, optionally substituted benzyl,

R₉ is H, alkyl of one to four carbons or optionally substituted benzyl;

R₁₀ is O or CH₂;

R₁₀ is H, OH, O-acyl of 14 carbons, O-alkyl of 1-4 carbons or anavailable electron;

A is N, CH or COH;

B is S, O, N, CH₂ or CH₂S;

a is 2 or 3;

b is 0 or 1;

m is an integer from 1 to 8;

n is 0 or 1;

p is 0, 1 or 2;

r is 2 or 3;

For each of X, Y and Z, each of the substituents shown is defined above.Also included is a compound of formula (I) where the ring containing theB and N moieties is fully reduced and contains no double bonds. It is tobe understood that for each of the formulas in this application,included are pharmaceutically-acceptable salts of the compoundrepresented by formula (I) and their individual stereoisomers andmixtures thereof. Preferred aspects are discussed hereinafter in theDetailed Description.

Another aspect of the invention is a compound of the formula (I)wherein:

R is N(OH)R₈;

each of R₁, R₂ and R₃ is H or CH₃;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

R₆ is H, OH, alkyl of 1-6 carbons or halogen;

R₈ is

R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

A is N or CH;

B is S, O, N, CH₂ or CH₂S;

n and p each is 0;

Z is

wherein each of the substituents shown is described above.

Another aspect of this invention is a compound of the formula (I)wherein:

R is OH, OR₇ or N(OH)R₈;

each of R₁, R₂ and R₃ is H or CH₃;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is (CH₂)_(a)(R₁₀)_(b)(CH₂)_(a)R₁₀(CH₂)_(a)(R₁₀)_(b)X;

R₆ is H, OH, alkyl of 1-6 carbons or halogen;

R₇ is alkyl of 1-4 carbons or optionally substituted benzyl;

R₈ is H, alkyl of 1-4 carbons, optionally substituted benzyl or(CH₂)_(m)N(OH)C(O)R₉

R₉ is H, alkyl of 1-4 carbons or optionally substituted benzyl.

R₁₀ is O or CH₂;

R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

A is N or CH;

B is S, O, N, CH₂ or CH₂S;

a is 2 or 3;

b is 0 or 1;

n and p each is 0;

r is 2 or 3; and

X is

wherein each of the substituents shown is described above,

Another aspect of this invention is a compound of the formula (I)wherein:

R is OH, OR₇ or N(OH)R₈;

each of R₁, R₂ and R₃ is H or CH₃;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

R₆ is (CH₂)_(a)R₁₀(CH₂)_(r)R₁₀Y;

R₇ is alkyl of 1-4 carbons or optionally substituted benzyl;

R₈ is H, alkyl of 1-4 carbons, optionally substituted benzyl or(CH₂)_(m)N(OH)C(O)R₉

R₉ is H, alkyl of 1-4 carbons or optionally substituted benzyl;

R₁₀ is O or CH₂;

R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

A is N or CH;

B is S, O, N, CH₂ or CH₂S;

a is 2 or 3;

b is 0 or 1;

n and p each is 0;

r is 2 or 3;

Y is

wherein each of the substituents shown is described above.

Another aspect of the invention is a compound of the formula:

wherein:

R₁ is H or CH₃;

R₂ is H or CH₃;

R₃ is H or CH₃;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 14 carbons or O-alkyl of 14 carbons;

R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

A is N or CH;

B is S, O, N, CH₂ or CH₂S;

a is 1, 2 or 3;

b is an integer from 2 to 8;

n is 0 or 1;

p is 0, 1 or 2;

r is 1, 2 or 3; and

s is 1, 2 or 3.

Another aspect of the invention is a compound of formula (I) wherein:

R is OH, OR₇ or N(OH)R₈;

R₁, R₂ and R₃ are H or CH₃;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

R₆ is hexyl;

R₇ is alkyl of 14 carbons or optionally substituted benzyl;

R₈ is H, alkyl of 14 carbons, optionally substituted benzyl or(CH₂)_(m)N(OH)C(O)R₉;

R₉ is H, alkyl of 1-4 carbons or optionally substituted benzyl;

R₁₁ is H, OH, O-acyl of 14 carbons or O-alkyl of 1-4 carbons;

A is N or CH;

B is S, O, N, CH₂ or CH₂S;

m is an integer from 1 to 8;

n is 0 or 1; and

p is 0, 1 or 2.

Another aspect of the invention is a method of treating malaria in ananimal, which method comprises administering an antimalarial amount ofcompound set forth in this summary of the invention.

Another aspect of the invention is a method of preparing a compositionuseful for treating malaria, which method comprises combining thecompound set forth above in this summary of the invention with apharmaceutically acceptable excipient.

Another aspect of the invention is a pharmaceutical composition designedfor treating malaria. The composition comprises a compound of formula(I) or formula (IV) in combination with a pharmaceutically acceptableexcipient.

Another aspect of the invention is an article of manufacture thatcomprises a pharmaceutical composition having a compound represented byformula (I) or (IV) with a pharmaceutically acceptable excipient inassociation with labeling describing the use of the composition fortreating malaria.

DETAILED DESCRIPTION AND PRESENTLY PREFERRED EMBODIMENTS

The present invention is based on the discovery that iron-chelatingcompounds of the above formulas are effective, i.a., against Plasmodiumfalciparum. Such compounds deprive this parasite of much needed iron forits metabolic processes. The compounds can be administered to humans indoses wholly unsuitable for chronic therapy due to the brief dosinginterval required for the treatment of malaria.

Applicant has identified compounds for the eradication of the Plasmodiumparasite that is targeted at the regulation of cellular iron metabolism.Iron, the fourth most abundant element in the earth's crust, is alsoubiquitous in all life forms. Eukaryotic microorganisms require iron tosustain life. Iron is critical for use by cytochromes and as a cofactorfor enzymes in electron-carrying proteins, for example.

While iron chelators, such as desferrithiocin (DFT) have been used forthe treatment of tranfusion-induced iron overload in β-thalassemia andaplastic anemia, such compounds were not contemplated for use in thetreatment of malaria prior to Applicant's invention. DFT is a superioriron chelating agent. It is well absorbed orally and is highly efficientat complexing with iron ions. However, it can be highly nephrotoxic whenused over time. This is particularly problematic, since chelationtherapy is a lifetime treatment of transfusion induced iron-overload inpatients suffering with β-thalassemia. The chronic toxicity of DFT hasresulted in its complete abandonment for treatment of chronic ironoverload, despite its high efficiency as an iron chelator.

It has been discovered that, in part because treatment of malaria withcompounds disclosed in this application would only require a briefdosing interval, patients would escape the chronic toxicity associatedwith the historical uses of DFT and related compounds. In fact, the newdimension of toxicity of siderophores for the treatment of malariapermits the development of new DFT analogs that are even more efficientiron chelators yet still provide manageable toxicity over the shortercourse of treatment for this disease. Thus, analogs of DFT that wouldnot be acceptable for traditional iron chelation therapy may becomeviable candidates for the treatment of malaria, particularly for deadlyP. falciparum malaria.

X-ray crystallography studies of the desferrithiocin pharmacophore haveindicated that the three ligating centers, i.e., the aromatic hydroxyl,the thiazoline nitrogen, and the carboxyl group, are important to thecompound's iron clearing capabilities. Any structural modifications tothe above functional groups should affect the ability of DFT tocoordinate with iron. An understanding of how to minimize the toxicityof analogs of DFT, however, has remained unclear. The present inventionprovides a range of compounds that strike a balance between optimal ironchelating ability and minimum toxcicity.

Compounds Useful in the Invention

“Alkyl” means a fully saturated hydrocarbon radical having the number ofcarbon atoms indicated. For example, alkyl of 1 to 6 includes, e.g.,methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl,arnyl, n-hexyl, and the like.

“Acyl” means a radical of the formula

where R is a hydrocarbon such as alkyl. An acyl of one to four carbonswould include those where R is an alkyl of one to three carbons. Thesewould include for example acetyl (CH₃—C(O)—), propionyl (CH₃—CH₂—C(O)—)or butyryl (CH₃(CH₂)₂C(O)—) or isobutyryl.

“Hexyl” means an alkyl of 6 carbons of any isomeric configuration, suchas n-hexyl, 1-methylpentyl, 1-ethylbutyl, 1,1-dimethylbutyl and thelike. The n-hexyl radical is preferred.

“Optionally substituted benzyl” is a benzyl group, i.e., phenylmethyl(PhCH₂—), that is either unsubstituted or substituted with one to fourcarbons, hydroxy, alkoxy of one to four carbons, halogen, acyl of one tofour carbons, and the like.

Compounds useful for preparing the composition and article ofmanufacture of this invention and for treating malaria are broadlydefined as compounds represented by Formulas (I) and (IV). Includedwithin the scope of the invention are the compounds per se,pharmaceutically acceptable salts of the compounds, and stereoisomeric(e.g. enantiomers, diastereomers) variations of the compounds. Formula Iis the following:

The substituents of formula (I) are defined as follows:

R is OH, OR₇, or N(OH)R₈;

R₁ is H, CH₃ or an available electron;

R₂ is H, CH₃ or an available electron;

R₃ is H, CH₃ at the (R) or (S) configuration, or an available electronand together with either-R₁ or R₂ when one is an available electron,forms a double bond with the R₁/R₂ carbon;

R₄ is H, acyl of 14 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 14 carbons, O-alkyl of 14 carbons, or

(CH₂)_(a)(R₁₀)_(b)(CH₂)_(a)R₁₀(CH₂)_(a)(R₁₀)_(b)X;

R₆ is H, OH, alkyl of 1-6 carbons, a halogen, (CH₂)_(a)R₁₀(CH₂)_(r)R₁₀Y,or is —C═C—C═C—, which, together with R₁₁ when R₁₁ is an availableelectron, forms a fused ring system as follows.

R₇ is alkyl of one to four carbons or optionally substituted benzyl;

R₈ is H, alkyl of one to four carbons, optionally substituted benzyl,

R₉ is H, alkyl of one to four carbons or optionally substituted benzyl;

R₁₀ is O or CH₂;

R₁₁ is H, OH, O-acyl of 1-4 carbons, O-alkyl of 14 carbons or anavailable electron;

A is N, CH or COH;

B is S, O, N, CH₂ or CH₂S;

a is 2 or 3;

b is 0 or 1;

m is an integer from 1 to 8;

n is 0 or 1;

p is 0, 1 or 2;

r is 2 or 3;

For each of X, Y, and Z, each of the substituents shown is definedabove.

Also included is a compound of formula (I) where the ring containing theB and N moieties is fully reduced and contains no double bonds. Thesecompounds can be subdivided into those where A is N (i.e. pyridylderivatives) and those where A is CH (i.e. benzene derivatives).

When A is N, those compounds where B is S and each of n and p is 0(thiazolines) are particularly useful. Preferred are compounds where

-   -   each of R₁, R₂ and R₃ is H or CH₃;    -   each of R₅ and R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of        1-4 carbons,    -   R₆ is H, OH, alkyl of 1-6 carbons or a halogen, and    -   R₇ is alkyl off 1-4 carbons.        Of this subgroup, compounds where R₄ is H and R is OH are        preferred. A representative compound is one where each of R₁,        R₂, R₃, R₅, R₆ and R₁₁ is H (compound 2 in Table I).        In the instance where R is N(OH)R₈, a compound where R₈ is        methyl is useful. A representative compound is one wherein each        of R₁, R₂, R₃, R₅, R₆ and R₁₁ is H (Compound 22 in Table I);        When R₈ is (CH₂)_(m)N(OH)COR₉, a representative compound is that        wherein each of R₁, R₂, R₃, R₅, R₆ and R₁₁ is H; m is 5; and R₉        is CH₃. (Compound 23 in Table 1); When R₈ is        (CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(OH)Z, a representative compound is one        where each of R₁, R₂, R₃, R₅, R₆ and R₁₁ is H. (Compound 25 in        Table I).

When A is CH, those compounds wherein B is S, and n and p each is 0(thiazolines) are particularly useful. Preferred is the subgroup wherein

-   -   each of R₁, R₂, R₃ and R₄ is H or CH₃,    -   each of R₅ and R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of        1-4 carbons,    -   R₆ is H, OH, alkyl of 1-6 carbons or a halogen, and    -   R₇ is alkyl of 1-4 carbons.        Of this subgroup, compounds where R₄ is H and R is OH are of        particular interest.        When R₁₁ is H, representative compounds include those wherein        each of R₁, R₂, R₃, R₅ and R₆ is H (Compound 3 in Table I);        wherein R₆ is OH and each of R₁, R₂, R₃ and R₅ is H (Compound 29        in Table I); wherein R₆ is F and each of R₁, R₂, R₃ and R₅ are H        (Compound 29a in Table I); and wherein R₆ is OH, R₃ is CH₃, and        R₁, R₂, and R₅ is H (Compound 33 in Table I). A representative        compound where R₁₁ is OH, is one wherein each of R₁, R₂, R₃, R₅        and R₆ is H. (Compound 5 in Table I). A compound where R₁₁ is        OH, R₆ is hexyl and each of R₁, R₂, R₃ and R₅ is H is also of        particular interest. Other compounds where R₁₁ is OH, include        one, wherein R₆ is OH and each of R₁, R₂, R₃ and R₅ is H.        (Compound 35 in Table I) and one, wherein R₆ is OH, R₃ is CH₃,        and each of R₁, R₂ and R₅ is H. (Compound 38 in Table I).

Compounds in which R₅ is(CH₂)_(a)(R₁₀)_(b)(CH₂)_(a)R₁₀(CH₂)_(a)(R₁₀)_(b)X, can be considered tobe a “dimer” in that there are two essentially similar parts of themolecule. A particularly interesting dimer is one wherein R₁₁ is OH, andeach of R₁ and R₂ is H, especially wherein a is 2 and each of R₃ and R₆is H. Representative compounds are those where R₁₀ is CH₂ and b is 0(Compound 40 in Table I); wherein R₁₀ is CH₂ and b is 1 (Compound 41 inTable I); wherein R₁₀ is O and b is 0 (Compound 42 in Table I); andwherein R₁₀ is O and b is 1 (Compound 43 in Table I).

Another “dimer” is represented by Formula (I), wherein R₆ is(CH₂)_(a)R₁₀(CH₂)_(r)R₁₀Y. Preferred in this dimer subgroup arecompounds and wherein R₁₁ is OH, and each of R₁ and R₂ is H and whereina is 3, and each of R₃ and R₅ is H. Of this preferred dimer subgroup,compounds of special interest are those wherein R₁₀ is CH₂ and r is 2(Compound 44 in Table I); wherein R₁₀ is CH₂ and r is 3 (Compound 45 inTable I); wherein R₁₀ is O and r is 2 (Compound 46 in Table I), andwherein R₁₀ is O and r is 3 (Compound 47 in Table I).

Of the general benzene/thiazoline compounds, those wherein R is N(OH)R₈(i.e. hydroxamates) are of significant interest. Of these hydroxamates,those dimers wherein R₆ is (CH₂)_(a)R₁₀(CH₂)_(r)R₁₀Y are of particularinterest, especially those wherein R₁₁ is OH, and each of R₁ and R₂ is Hand wherein a is 3 and each of R₃ and R₅ is H. A representative compoundis one wherein R₈ is CH₃, R₁₀ is O and r is 3. (Compound 48 in Table I).Another representative hydroxamate dimer is one wherein R₈ is(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂N(OH)Z. The dimers wherein R₁₁ is OH and each ofR₁ and R₂ is H are of significant interest. A representative compound isone wherein each of R₃, R₅ and R₆ is H (Compound 50 in Table I).

Another useful compound is one of formula (IV), namely.

In formula (IV), the substituents are defined as follows:

R₁ is H or CH₃;

R₂ is H or CH₃;

R₃ is H or CH₃;

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

R₁₁ is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons;

A is N or CH;

B is S, O, N, CH₂ or CH₂S;

a is 1, 2 or 3;

b is an integer from 2 to 8;

n is 0 or 1;

p is 0, 1 or 2;

r is 1, 2 or 3; and

s is 1, 2 or 3.

Those compounds of particular interest are those wherein A is CH,especially wherein B is S with R₄ being H and further wherein R₁₁ is OH,R₁, R₂ and R₅ are H, and each of n and p is 0. A representative compoundis one wherein each of r and s is 3, a is 2, and b is 2 (Compound 49 inTable I).

Compounds that are particularly useful in the composition, the method oftreatment and the article of manufacture of this invention are set forthin Table I. In Table I, the left hand, vertical column lists thesubstituent of formula (I), (i.e. A, R₁, a, etc) while the top,horizontal row gives the compound number (i.e. 1-51). The compounddesignated as #8 is of particular interest.

TABLE I Cpd 1 2 3 4 5 6 7 8 9 11 12 14 15 16 17 A N N CH CH CH CH CH CHCH CH CH N CH CH CH B S S S S S S S S S N CH₂ CH₂S S S S R OH OH OH OHOH OH OH OH OH OH OH OH OH OH OH R1 H H H H H H H CH₃ H H H H H H H R2 HH H H H H H CH₃ H H H H H H H R3 CH₃ H H H H H H H CH₃ H H H H H H R4 HH H H H H H H H H H H H H H R5 H H H OH H OCH₃ H H H H H H H H H R6 H HH H H H H H H H CH₃ H H H H R7 — — — — — — — — — — — — — — — R8 — — — —— — — — — — — — — — — R9 — — — — — — — — — — — — — — — R10 — — — — — — —— — — — — — — — R11 H H H H OH H CO₂H H H H H H H H H a — — — — — — — —— — — — — — — b — — — — — — — — — — — — — — — m — — — — — — — — — — — —— — — n 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 p 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 r— — — — — — — — — — — — — — — Cpd 18, 19 20, 21 22 23 24 25 28 29 29a 33A CH N N N N N CH CH CH CH B S S S S S S S S S S R OH OH N(OH)R₈ N(OH)R₈N(OH)R₈ N(OH)R₈ OH OH OH OH R1 H H H H H H H H H H R2 H H H H H H H H HH R3 H H H H H H CH₃ H H CH₃ R4 H H H H H H H H H H R5 H H H H H H H H HH R6 —C═C—C═C— —C═C—C═C— H H H H H OH F OH R7 — — — — — — — — — — R8 — —CH₃ (CH₂)_(m)NOHCOR₉ benzyl Z monomer — — — — (1) R9 — — — CH₃ — — — — —— R10 — — — — — — — — — — R11 bond bond H H H H OH H H H a — — — — — — —— — — b — — — — — — — — — — m — — — 5 — — — — — — n 0 0 0 0 0 0 0 0 0 0p 0 0 0 0 0 0 0 0 0 0 r — — — — — — — — — — Cpd 35 38 40 41 42 43 44 45A CH CH CH CH CH CH CH CH B S S S S S S S S R OH OH OH OH OH OH OH OH R1H H H H H H H H R2 H H H H H H H H R3 H CH₃ H H H H H H R4 H H H H H H HH R5 H H X monomer (2) X monomer (2) X monomer (2) X monomer (2) H H R6OH OH H H H H Y monomer Y monomer R7 — — — — — — — — R8 — — — — — — — —R9 — — — — — — — — R10 — — CH₂ CH₂ O O CH₂ CH₂ R11 OH OH OH OH OH OH OHOH a — — 2 2 2 2 3 3 b — — 0 1 0 1 0 0 m — — — — — — — — n 0 0 0 0 0 0 00 p 0 0 0 0 0 0 0 0 r — — — — — — 2 3 Cpd 46 47 48 50 51 A CH CH CH CHCH B S S S S S R OH OH N(OH)R₈ (4) N(OH)R₈ OH R1 H H H H H R2 H H H H HR3 H H H H H R4 H H H H H R5 H H H H H R6 Y monomer (3) Y monomer (3) Ymonomer (4) H hexyl R7 — — — — — R8 — — CH₃ Z monomer (5) — R9 — — — — —R10 O O O — — R11 OH OH OH OH OH a 3 3 3 — — b 0 0 0 — — m — — — — — n 00 0 0 0 p 0 0 0 0 0 r 2 3 3 — — (1) R₈ = (CH₂)₂—O—(CH₂)₂—O(CH₂)₂—Z whereA is CH, B is S, each of R₁₋₆ is H, R₁₁ is H, n is 0 and p is 0 (2) R₅ =(CH₂)_(a)(R₁₀)_(b)(CH₂)_(a)R₁₀(CH₂)_(a)(R₁₀)_(b)X, where A is CH, B isS, R is OH, each of R₁₋₄ is H, R₆ is H, R₁₁ is OH, n is 0 and p is 0 (3)R₆ = (CH₂)_(a)R₁₀(CH₂)_(r)R₁₀Y, where A is CH, B is S, R is OH, each ofR₁₋₅ is H, R₆ is H, n is 0 and p is 0 (4) See Formula (IV) (5) R₈ =(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—Z where A is CH, B is S, each of R₁₋₆ is H, nis 0 and p is 0

As pointed out hereinbefore, the compounds useful in this inventioninclude all the stereochemical modifications. The compounds of formula(I) are characterized by at least one asymmetric carbon atom marked withan asterisk (*) (Another possible chiral atom is the R₁/R₂ carbon). Thebonds-surrounding these carbon atoms are arranged tetrahedrally, and thesubstituents thus bonded to the asymmetric carbon atoms are in fixedpositions. The compounds of formula (I) represent optical enantiomersexhibiting either the (S) or (R) configuration as shown in (i) and (ii)below, respectively:

For purpose of this application, the R₃ and the (CH₂)_(p)C(O)Rsubstituents will be designated as

This designation is to be interpreted encompassing as the (R) and the(S) configurations, as well as racemic modifications.

Compounds useful in this invention are of either the (S) or (R)configuration, with the (S) enantiomer of formula (I) being preferred. Aparticular configuration can be specified according to the procedureproposed by R. S. Cahn, Sir Christopher Ingold and V. Prelog. See forexample “Organic Chemistry,” 3^(rd) Edition, by R. T. Morrison and R NBoyd at pages 130-133. If compounds of formula (I) contain two chiralcenters, e.g. when R1 and R2 are different, these compounds can beconsidered diastereomers if the stereoisomer is not a mirror image ofanother stereoisomer. Thus the individual enantiomers, diastereomers,racemic modifications, and mixtures are also within the scope of theinvention and claims.

The invention also includes pharmaceutically-acceptable salts of thecompounds of formulas (I) and (IV), particularly the carboxylic acids offormula (I). Such salts include, for example, anunonium salts and metalsalts such as the alkali metal and alkaline earth metals salts, e.g.,sodium, potassium, magnesium or calcium salts, as well as divalent metalsalts such as zinc. Salts with suitable organic amines are alsoincluded, e.g., aliphatic, cycloaliphatic, cycloaliphatic-aliphatic oraraliphatic primary, secondary or tertiary mono-, di- or poly-amines,and also heterocyclic bases. Such amines are, for example, loweralkylamines, for example, triethylamine, hydroxy-lower alkyl-amines, forexample, 2-hydroxyethylamine, bis-(2-hydroxy-ethyl)-amine ortris-(2-hydroxyethyl)-amine, basic aliphatic esters of carboxylic acids,for example, 4-aminobenzoic acid 2-diethylaminoethyl ester, loweralkyleneamines, for example, 1-ethylpiperidine, cycloalkylamines, forexample, dicyclo-hexylamine, or benzylamines, for, example,N,N′-dibenzyl-ethylenediamine, also bases of the pyridine type, forexample, pyridine, colladine or quinoline. Further salts includeinternal salts (zwitterionic forms of compounds of the invention),wherein a basic group, for example, the basic nitrogen atom present inthe pyridine ring, is protonated by a hydrogen ion originating from anacid group in the molecule.

Preparation of Compounds

The compounds useful in this invention are prepared in accordance withprocedures known in the art or ascertainable therefrom or in accordancewith procedure guidelines set forth in this application. For example,the following patent and laid-open applications are useful and areincorporated herein by reference: U.S. Pat. No. 4,406,905, PCTInternational Publication #WO 94/11367, and PCT InternationalPublication #WO 97/36885

Certain compounds are known and useful in the preparation of othercompounds useful in this invention. These include

(1) (S)-desmethyldesferrithiocin,

(2) (S)-desmethyldesferrithiocin, N-methylhydroxamate

In general, useful compounds where B is S, n is 0, and p is 0 areprepared by reacting a compound of formula (II) with a compound offormula (III). Formula (II) is

where

R₄ is H, acyl of 1-4 carbons or alkyl of 1-4 carbons;

R₅ is H, OH, O-acyl of 1-4 carbons, or O-alkyl of 1-4 carbons;

R₆ is H, OH, alkyl of 1-6 carbons, a halogen, or is —C═C—C═C—, which,together with R₁₁ when R₁₁ is an available electron, forms a fused ringsystem as follows:

R₁₁ is H, OH, O-acyl of 1-4 carbons, O-alkyl of 1-4 carbons or anavailable electron; A is N, CH or COH; and W is carboxy or a reactivefunctional derivative of a carboxy group. Formula (III) is

where each of R₁, R₂ and R₃ is H or CH₃. Hydroxy groups are optionallyprotected to produce a desired compound after splitting off optionallypresent protective groups and, optionally, conversion to a suitable saltor hydroxamate. An exemplary discussion of how to make compounds usefulin this invention is found in U.S. Pat. No. 4,406,905, issued to Zähner,et al. on Sep. 27, 1983. This patent is incorporated herein byreference. Other discussions regarding how to make compounds useful inthis invention are set forth in PCT International Publication NumberWO94/11367 and WO97/36885. These too (along with any corresponding U.S.counterparts) are incorporated herein by reference.

Free hydroxy groups present in the compounds of the above formulas areoptionally protected by conventional protecting groups. Such protectinggroups protect the hydroxy groups from undesired condensation reactions,substitution reactions and the like. The protecting groups can beintroduced and removed easily, i.e., without undesirable secondaryreactions taking place, for example, by solvolysis or reduction, in amanner known per se. Protecting groups and the methods by which they areintroduced and split off are described, for example, in “ProtectiveGroups in Organic Chemistry,” Plenum Press, London, N.Y. (1973) and alsoin “Methoden der organischen Chemie,” Houben-Weyl, 4^(th) edition, Vol.15/1, Georg Thieme Verlag, Stuttgart (1974).

Suitable hydroxy-protecting groups are, for example, acyl radicals suchas lower alkanoyl optionally substituted, for example, by halogen suchas 2,2-dichloroacetyl, or acyl radicals of carbonic acid semiesters,especially t-butoxy-carbonyl, optionally substituted benzyloxycarbonyl,for example, 4-nitrobenzyloxycarbonyl, or 2-halo-lower alkoxycarbonylsuch as 2,2,2-trichloroethoxycarbonyl, also trityl or formyl, or organicsilyl radicals, also etherifying groups that can readily be split offsuch as t-lower alkyl, for example, t-butyl, or 2-oxa- or2-thia-cycloalkyl having 5 or 6 ring atoms, for example, tetrahydrofurylor 2-tetrahydropyranyl or corresponding thia analogs, and alsooptionally substituted 1-phenyl-lower alkyl such as optionallysubstituted benzyl or diphenylmethyl, there coming into consideration assubstituents of the phenyl radicals, for example, halogen such aschlorine, lower alkoxy such as methoxy, and/or nitro.

A reactive functional derivative of a carboxy group W, above, is, forexample, an acid anhydride, an activated ester or an activated amide,cyano, a group of the formula —C(OR_(a))₃ or —C(═NH)—R_(a) in whichR_(a) is lower alkyl. Corresponding derivatives are known in the art.

Of the anhydrides, the mixed anhydrides are especially suitable. Mixedanhydrides are, for example, those with inorganic acids such ashydrohalic acids, i.e., the corresponding acid halides, for example,chlorides or bromides, also with hydrazoic acid, i.e., the correspondingacid azides. Further mixed anhydrides are, for example, those withorganic carboxylic acids such as with lower alkanecarboxylic acidsoptionally substituted, for example, by halogen such as fluorine orchlorine, for example, pivalic acid or trichloroacetic acid, or withsemiesters, especially lower alkyl semniesters of carbonic acid such asthe ethyl or isobutyl semniester of carbonic acid, or with organic,especially aliphatic or aromatic, sulfonic acids, for example,p-toluenesulfonic acid. Of the activated esters, there may be mentioned,for example, esters with vinylogous alcohols (i.e., enols such asvinylogous lower alkenols), or iminomethyl ester halides such asdimethyliminomethyl ester chloride (prepared from the carboxylic acidand, for example, dimethyl-(1-chloroethylidine)-iminiurn chloride of theformula (CH₃)₂N^(⊕)═C(Cl)CH₃Cl⁻, which can be obtained, for example,from N,N-dimethylacetamide and phosgene), or aryl esters such aspreferably suitable substituted phenyl esters, for example, phenyl estersubstituted by halogen such as chlorine, and/or by nitro, for example,4-nitro-phenyl ester, 2,3-dinitrophenyl ester or2,3,4,5,6-penta-chlorophenyl ester, N-hetero-aromatic esters such asN-benztriazole esters, for example, 1-benztriazole ester, orN-diacylimino esters such as N-succinylamino or N-phthalylimino ester.Suitable activated amides are, for example, imidazolides, also1,2,4-trazolides, tetrazolides or 1,2,4-oxadiazolinonides.

The activation of the carboxy group W, above, in the compounds of theabove formula can also be effected in situ.

A reactive derivative of a cysteine-related compound of the aboveformulas is a compound in which the amino and/or mercapto group isactivated for the reaction with the carboxy group of a compound of theabove formulas, that is to say, is present in nucleophilic form. Theamino group is activated, for example, by reaction with a phosphite.

The reaction of the above compounds in which W represents carboxy withthe cysteine derivative is preferably carried out in the presence of asuitable condensation agent or under dehydrating conditions, forexample, while removing the water of reaction by azeotropicdistillation. Customary condensation agents are, for example,carbodiimides, for example, N,N′-diethyl-, N,N′-dipropyl-,N,N′-dicyclohexyl- or N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide,suitable carbonyl compounds, for example, carbonyldiimidazole, or1,2-oxazolium compounds, for example,2-ethyl-5-phenyl-1,2-oxazolium-3′-sulfomate or2-tert.-butyl-5-methyl-isoxazolium perchlorate, for a suitable acylaminocompound, for example, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline,furthermore diphenylphosphoryl azide. The condensation reaction iscarried out preferably in an anhydrous reaction medium, preferably inthe presence of a solvent or diluent, for example, methylene chloride,benzene or tetrahydrofuran and, if necessary, while cooling or heating,for example, at ambient temperature or at slightly elevated temperature,and/or in an insert gas atmosphere. If a compound in which W representsan acid anhydride derivative of a carboxy group is carried out, thereaction is performed under essentially the same conditions in thepresence of a basic agent such as the sodium or potassium salt orcarbonic acid, or a tertiary amino compound such as a tri-C₁-C₄-alkylamine, for example, triethylamine, or a pyridine base such as pyridineor quinoline.

A preferred form of this process according to the invention is thereaction of a compound of the above formulas in which W represents cyanowith a cysteine derivative of the above formula. The reaction is carriedout in an inert solvent such as an aqueous solvent at ambienttemperature or, preferably, at slightly elevated temperature, forexample, at about 50° to 80° C., and preferably under an inert gasatmosphere. The carboxylic acid (or a reactive functional derivativethereof) is reacted with a compound of the formula

or with a compound which is convertible thereto. A preferred functionalderivative of a carboxy group according to the invention is theN-succinylimino ester. The reaction is performed in an inert solventsuch as an aprotic solvent, for example, dimethylformamide,dimethylsulfoxide or dioxane or a C₁-C₄ alkanol such as methanol, atambient temperature or while cooling, for example, at about 0° C.

In resulting compounds in which one or more functional (hydroxy) groupsare protected, the latter can be freed, optionally in stages orsimultaneously, in a manner know per se, by means of solvolysis,especially hydrolysis or acidolysis, or in some cases also by means ofcareful reduction. Silyl protecting groups are advantageously split offwith fluorides, for example, tetraethylammonium fluoride.

Salts of compounds of the invention can be manufactured in a mannerknown per se. Thus, salts of compounds having acidic groups can beformed, for example, by treating with metal compounds such as alkalimetal salts of suitable organic carboxylic acids, for example, thesodium salt of α-ethylcaproic acid, or with inorganic alkali metal oralkaline earth metal salts, for example, sodium bicarbonate, or withammonia or a suitable organic amine, preferably stoichiometricquantities or only a small excess of the salt-forming agent being used.Acid addition salts of compounds of the invention are obtained in acustomary manner, for example, by treating with an acid or a suitableanion-exchange reagent. Internal salts of compounds of the invention(zwitterionic forms) can be formed, for example, by neutralizing thecompounds or salts such as acid addition salts, to the isoelectricpoint, for example, with weak bases, or by treating with liquid ionexchangers.

Salts can be converted in a customary manner into the free compounds:metal and ammonium salts can be converted into the free compounds, forexample, by treating with suitable acids, and acid addition salts, forexample, by treating with a suitable basic agent.

The starting materials are available commercially and/or known or can bemanufactured by known processes.

The racemate can be split in a manner known per se, for example, afterconversion of the optical antipodes into diastereoisomers, for example,by reaction with optically active acids or bases.

Where a compound is a carboxylic acid, a racemic mixture of a carboxylicacid may be resolved by first treating the racemate with an opticallyactive amine base to form a mixture of diastereomeric salts. Examples ofoptically active amine bases that may be used for this purpose are(R)-(+)-∝-methylbenzylamine, (s)-(−)-∝-methylbenzylamine,(1R,2S)-(−)-ephedrine, quinine, and quinidine. The thusly formeddiastereomeric salts have different properties, such as solubility, andthe diastereomers may therefore be separated by selectiverecrystallization from a suitable solvent. The optically activecarboxylic acids may then be obtained by re-acidification of theseparated diastereomeric salts.

Alternatively, a racemic mixture of a carboxylic acid may be treatedwith an optically active alcohol to form a mixture of diastereomericesters. Examples of optically active alcohols that may be used for thispurpose are (1R,2S,5R)-(−)-menthol, (1S,2R,5S)-(+)-menthol,(R)-(−)-2-octanol, and (S)-(+)-2-octanol. The thusly-formed mixture ofdiastereomeric esters may then be separated by chromatography. Theoptically active carboxylic acids may then be obtained from theseparated diastereomeric esters by conventional techniques, such astreatment of the esters with sodium hydroxide or lithium hydroxidefollowed by reacidification.

If a compound is an ester, a racemate of an ester may be resolved intothe enantiomers by first resolving a racemic mixture of thecorresponding carboxylic acid using one of the methods described above.The optically active ester may be obtained by esterification of thecorresponding optically active carboxylic acid by procedures similar tothose used to prepare a racemic ester.

Alternatively, a racemic mixture of a carboxylic acid or a racemicmixture of an ester may be separated into the individual enantiomers byhigh performance liquid chromatography using a suitable chiralstationary phase and a suitable eluent.

Further discussion of reaction parameters for compounds of this type canbe found in articles by Bergeron et al. J. Med. Chem. 1994, 37,1411-1417; J. Med. Chem. 1999, 42, 95-108; J. Med. Chem. 1999, 42,2432-2440 and in U.S. Ser. Nos. 08/624,289 and 08/532,805, both of whichare incorporated herein by reference.

Pharmaceutical Preparations

The compounds described above are useful for treating malaria and thusare useful for the manufacture of pharmaceutical compositions whichcontain an effective amount of the active substance in admixture withinorganic or organic, solid or liquid, pharmaceutically acceptablecarriers. Thus, another aspect of this invention is an antimalarialcomposition of a compound described herein in combination with apharmaceutically acceptable excipient.

The pharmaceutical compositions according to the invention are thosewhich are suitable for enteral, such as oral, administration and forparenteral, such as subcutaneous or intravenous, administration tohumans, and which contain the pharmacological active substance togetherwith a pharmaceutically acceptable carrier. The dosage of the activesubstance depends on various factors such as the age, weight, severityof the malarial condition, and other factors a doctor might identify.

The novel pharmaceutical preparations contain from approximately 10% toapproximately 95%, and preferably from approximately 20% toapproximately 90%, of the active substance. Pharmaceutical compositionsaccording to the invention can, for example, be in unit dose form, suchas dragées, tablets, capsules, suppositories or ampoules, and containfrom approximately 0.1 g to approximately 2.0 g, and preferably fromapproximately 0.3 g to approximately 1.0 g, of the active ingredient.

The pharmaceutical compositions of the present invention aremanufactured in a manner known per se, for example, by means ofconventional mixing, granulating, confectioning, dissolving orlyophilizing processes. Pharmaceutical compositions for oral use can beobtained by combining the active substance with one or more solidcarriers, if desired, granulating a resulting mixture and processing themixture or granulate, if desired or necessary after the addition ofsuitable adjuncts, to form tablets or dragee cores. In so doing, theycan also be incorporated into plastics carriers which release the activesubstances or allow them to diffuse in controlled amounts.

Suitable carriers are especially fillers such as sugars, for example,lactose, saccharose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example, tricalcium phosphate or calciumhydrogen phosphate, also binders such as starches, for example, corn,wheat, rice or potato starch, gelatine, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, and/or, if desired, disintegrators such as theabove-mentioned starches, also carboxymethyl starch, cross-linkedpolyvinylpyrorrolidone, agar, alginic acid or a salt thereof such assodium alginate. Adjuncts are especially flow-regulating and lubricatingagents, for example, silica, talc, stearic acid or salts thereof such asmagnesium or calcium stearate, and/or polyethylene glycol. Dragée coresare provided with suitable coatings that are, if desired, resistant togastric juice, there being used, inter alia, concentrated sugarsolutions which optionally contain gum arabic, talc,polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,lacquer solutions in suitable organic solvents or solvent mixtures or,for the manufacture of coatings that are resistant to gastric juice,solutions of suitable cellulose preparations such as acetylcellulosephthalate or hydroxypropylmethylcellulose phthalate. Coloring substancesor pigments can be added to the tablets or dragée coatings, for example,for the purpose of identification or for indicating different doses ofactive substance.

Other orally administrable pharmaceutical compositions are dry-filledcapsules made of gelatin and also soft, sealed capsules made of gelatinand a plasticizer such as glycerol or sorbitol. The dry-filled capsulesmay contain the active ingredient in the form of a granulate, forexample, in admixture with fillers such as corn starch, binders and/orglidants such as talc or magnesium stearate and optionally stabilizers.In soft capsules, the active ingredient is preferably dissolved orsuspended in suitable liquids or wax-like substances such as fatty oils,paraffin oil or polyethylene glycols, it being possible also forstabilizers to be added.

Other forms of oral administration are, for example, syrups prepared ina customary manner that contain the active ingredient in, for example,suspended form and in a concentration of approximately from 5% to 20%,and preferably approximately 10%, or in a similar concentration thatprovides a suitable single dose when administered, for example, inmeasures of 5 or 10 ml. Also suitable are, for example, powdered orliquid concentrates for preparing shakes, for example, in milk. Suchconcentrates can also be packed in single-dose quantities.

Particularly suitable dosage forms for parenteral administration aresterile aqueous solutions of an active ingredient in water-soluble form,for example, a water-soluble salt, or sterile aqueous injectionsuspensions which contain substances increasing the viscosity, forexample, sodium, carboxymethyl cellulose, sorbitol and/or dextran, andoptionally stabilizers. In addition, the active ingredient, with orwithout adjuvants, can also be in lyophilized form and brought intosolution prior to parenteral administration by the addition of suitablesolvents.

Generally, an injectable composition of the invention may be

-   -   1. a solution that is ready for injection, or    -   2. a dry soluble composition that is ready to be combined with a        solvent just prior to use,    -   3. or a liquid concentrate ready for dilution prior to        administration.        In preparing a composition for injection strict attention must        be paid to tonicity adjustment to avoid irritation.

The vehicle normally has no therapeutic activity and is nontoxic, butpresents the active constituent to the body tissues in a formappropriate for absorption. Absorption normally will occur most rapidlyand completely when the compound is presented as an aqueous solution.However, modification of the vehicle with water-miscible liquids orsubstitution with water-immiscible liquids can affect the rate ofabsorption. Preferably, the vehicle of greatest value for subcutaneouscomposition is water that meets the USP specification for water forinjection. Generally, water of suitable quality for compounding willeither be prepared by distillation or reverse osmosis to meet these USPspecifications. The appropriate specifications are spelled out inRemington: The Science and Practice of Pharmacy” 19^(th) Ed. At pps.1526-1528. In preparing the compositions which are suitable forsubcutaneous injection, one can use aqueous vehicles, water-misciblevehicles, and nonaqueous vehicles. Certain aqueous vehicles arerecognized officially because of their valid use in parenteralsgenerally.

Water-miscible vehicles are also useful in the formulation of theparenteral composition of this invention. These solvents are usedprimarily to affect the solubility of the compound. The most importantsolvents in this group are ethyl alcohol, polyethylene glycol andpropylene glycol.

These vehicles include fixed oils, for example, those of a vegetableorigin to allow for proper metabolism. For a USP subcutaneous injectioninjectable composition, the USP specifies limits for the degree ofunsaturation and free fatty acid content. The oils most commonly usedare corn oil, cottonseed oil, peanut oil, and sesame oil. Also usefulare certain, more recently developed neutral oils that are esters ofmedium-chain fatty acids, and are also call fractionated coconut oil.Medium chain fatty acids, i.e., those of about 8 to 10 carbon atoms,include compounds referred to as MIGLYOL®, which are sold by DynamitNobel. Five types of MIGLYOL® identified as MIGLYOL® 810, 812, 828, 829,and 840 are useful. These are more fully described in trade literaturefrom Dynamit Nobel. Certain other esters are also useful as nonaqueousvehicles, for example, triglycerides, propylene glycol diesters, and thelike.

Additional substances may be included in the injectable compositions ofthis invention to improve or safeguard the quality of the composition.Thus, an added substance may affect solubility, provide for patientcomfort, enhance the chemical stability, or protect preparation againstthe growth of microorganisms. Thus, the composition may include anappropriate solublizer, substances to make a solution isotonic,substances to act as antioxidants, and substances that act as apreservative to prevent the growth of microorganisms. These substanceswill be present in an amount that is appropriate for their function, butwill not adversely affect the action of the composition as a treatmentfor malaria. Examples of appropriate antimicrobial agents includethimerasol, benzethonium chloride, benzalkoniumchloride, phenol, methylp-hydroxybenzoate and propyl p-hydrodxybenzoate. Appropriate buffers andantioxidants may be found in “Remingtons” at p. 1529.

Generally, the sterile, parenterally injectable composition of thisinvention will comprise about 0.1% by wt. to about 50% by wt. of thecompound with the remainder being the appropriate excipient orexcipients.

Article of Manufacture

Another aspect of this invention is an article of manufacture thatcomprises an antimalarial composition comprising a compound representedby formula (I) or formula (IV), in combination with a pharmaceuticallyacceptable excipient, the article of manufacture further comprisingwritten instructions for administering the antimalarial composition to ahuman in a quantity sufficient to treat the malaria over time. This isan important aspect of the invention in that before a compound can beapproved for any particular use, it must be approved for marketing bythe United States Food and Drug Administration. Part of that processincludes providing a label that will accompany the pharmaceuticalcomposition which is ultimately sold. While the label will include adefinition of the composition and such other items such as the clinicalpharmacology, mechanism of action, drug resistance, pharmacokinetics,absorption, bioavailability, contraindications and the like, it willalso provide the necessary dosage, administration, and use, as discussedabove. Thus, the combination of the drug with appropriate labelinginstructions is important for the proper usage of the drug once it getson the market.

Treatment of Malaria

The term “treatment” as used herein covers any treatment of a diseasetreatable by an iron chelator in a mammal, particularly a human, andincludes:

(i) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(ii) inhibiting the disease, i.e. arresting its development; or

(iii) relieving the disease, i.e. causing regression of the disease.

Malaria is a disease that strikes various mammals, particularly humansand non-human primates, as well as certain birds. The compounds andcompositions of this invention may be used to treat malaria in mammalsand birds. Its primary application will be in treating humans in which amalignant form of malaria is caused by the protozoan known as Plasmodiumfalciparum, although the compounds may be used also to treat malariacaused by more benign protozoans, such as P. vivax, P. malariae, and P.ovale. The compounds are delivered at a level and for a time thatdeprives the parasite of iron for its metabolic processes, thus causingdeath of the parasites.

In general, the compounds of the invention may be administeredenterally, that is orally, or parenterally (i.e. intraperitoneally—IP;intramuscularly—IM; subcutaneously—SC; intravenously—IV; etc.), such asby single injection or infusion by IV. By subcutaneous administration itis meant that the drug in the form of an appropriate injectablecomposition is injected into the areola connective tissue just below theskin. The injection may be a solution, a suspension, or a formulationthat provides a controlled release of the active entity. Generally, thesubcutaneous administration will be done with excipients that aresuitable for subcutaneous administration, which means that theexcipients will have to meet USP considerations in being appropriate forinjectable compositions. Thus, the composition will need to be sterileto avoid any complications due to insterility at the injection site. Theparticular mode of treatment will depend on the severity of the disease,the age of the patient, the size of the patient, the relative health ofthe patient, and other factors of which the treating physician will beaware. The amount of the active ingredient that will be present in thecomposition to be injected and that will be injected is atherapeutically-effective amount, that is, an amount which is sufficientto result in successful treatment as defined above when administered toan animal exhibiting signs and symptoms of malaria. The therapeuticallyeffective amount will vary depending on the subject, the severity of theaffliction and the manner of administration, and may be determinedroutinely by one of ordinary skill in the art in light of the disclosureof this specification. For example, if a patient is extremely ill and isunable to ingest any food, then it may be necessary to provide a singleinjection or an IV infusion over a period that may be up to 72 hours.While in some cases, treatment could last up to a month, usually thetreatment regimen will last no more than two weeks, preferably less thanone week. The patient will be monitored by indications that the signsand symptoms are improving.

Suitable doses are in the general range from about 1 to about 250 mg/Kgbody weight of the recipient per day, preferably in the range from about5 to about 150 mg/Kg.

The following examples are given to teach one of ordinary skill in theart how to make compounds useful in this invention. The numericalreference to a compound refers to the compounds set forth in Table I ofthis application. The designation of formula A, B, C, etc. refers to thereaction sequence or scheme accompanying the example. DFT refers todesferrithiocin.

Example 1 Synthesis of Compounds 2, 3, 5-7, 9, 14-17 and 22-24

Compounds 2, 3, 5-7, 9 and 14-17 with the general formula C, weresynthesized by cyclocondensation of an o-hydroxyaryl nitrile A with acysteine derivative B (Scheme 1) (Bergeron et al., J. Med. Chem. 34:2072-2078 (1991); Bergeron et al., J. Med. Chem. 37:

1411-1417 (1994); Bergeron et al., J. Med. Chem. 42: 2432-2440 (1999);

(S)- and (R)-DesmethylDFTs 2 were synthesized by condensation of2-cyano-3-hydroxypyridine, obtained from 3-hydroxypyridine-N-oxide, withD- or L-cysteine, respectively, in pH6 phosphate buffer and methanol.Reaction of 2-hydroxypyridine with D- or L-cysteine provided (S)- and(R)-desazadesmethylDFTs 3, respectively.

The production of (S)-desazaDFT (9), identical to the natural product4-methylaeruginoic acid (Ryoo et al., J. Antibiot. 50: 256-258 (1997)),and 4′-hydroxydesazaDFT (28) was accomplished by cyclocondensation of2-cyanophenol or 2,4-dihydroxybenzonitrile, respectively, with(S)-α-methyl cysteine in buffered aqueous CH₃OH. The latter cyanocompound was prepared by treatment of 2,4-dihydroxybenzaldehyde withnitroethane in sodium acetate and acetic acid. Hydrolysis of DFT (1) in6 N HCl generated the unusual amino acid (S)-α-methyl cysteine.

Tridentate chelators 15-17, homologues of (S)-3 with a spacer betweenthe ligating centers, were synthesized as follows.(S)-4,5-Dihydro-2-(2-hydroxyphenylmethyl)-4-thiazolecarboxylic acid (15)was assembled by heating D-cysteine with 2-hydroxyphenylacetonitrile inmethanolic phosphate buffer.(S)-4,5-Dihydro-2-(2-hydroxyphenyl)-4-thiazoleacetic acid (16) and(S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolepropanoic acid (17) weremade by reacting (S)-3-amino-4-mercaptobutanoic acid or(S)-4-amino-5-mercaptopentanoic acid, respectively, and 2-cyanophenol inmethanolic phosphate buffer. The β- or γ-amino acid was in turn preparedfrom partially protected L-aspartic and L-glutamic acid (Chauvel et al.,J. Med. Chem. 37: 1339-1346 (1994); Wilk et al., Neuropeptides 16:163-168 (1990)).

(R)- and (S)-4,5-Dihydro-2-(2-,4-dihydroxyphenyl)-4-thiazolecarboxylicacid (5) were constructed by condensing 2,4-dihydroxybenzonitrile withL- or D-cysteine, respectively, in phosphate buffer and methanol.

The key step of the synthesis of(S)-4,5-dihydro-2-(2-hydroxy-3-methoxyphenyl)-4-thiazolecarboxylic acid(6) was cyclocondensation of D-cysteine with2-hydroxy-3-methoxybenzonitrile. The cyano intermediate was obtainedfrom o-vanillin by treatment with nitroethane in sodium acetate andacetic acid.(S)-4,5-Dihydro-2-(2-hydroxy-4-carboxyphenyl)-4-thiazolecarboxylic acid(7) was synthesized by a similar reaction sequence.4-Formyl-3-hydroxybenzoic acid was converted to 4-cyano-3-hydroxybenzoicacid using nitroethane in sodium acetate and acetic acid; cyclizationwith D-cysteine completed the synthesis of dicarboxylic acid chelator 7.

Condensation of 2-cyano-3-4-hydroxypyridine with DL-homocysteineafforded racemic2-(3-hydroxy-2-pyridinyl)₄H-5,6-dihydro-1,3-thiazine-4-carboxylic acid(14), a six-membered analogue of 2 (Scheme 1) (Bergeron et al., J. Med.Chem. 37: 1411-1417 (1994)).

Example 2 Synthesis of Compounds 18-20

Fused ring DFT analogue J, specifically compounds 18-21, weresynthesized by cyclization of an enantiomer of cysteine B (R₃=H, B=S,p=0) with an o-hydroxynaphthyl or -quinolinyl nitrile I (Scheme 2)(Bergeron et al., J. Med. Chem. 39: 1575-1581 (1996)).

Example 3 Synthesis of Compounds 22-24

Conversion of the carboxylic acid group of (S)-desmethyldesferrithiocin(DMDFT, 2), to an N-methylhydroxamate or to the pentacoordinatedihydroxamate compound resulted in compounds 22 and 23, respectively(Bergeron, et al., J. Med. Chem. 37:1411-1417 (1994)).

Compound 24, the N-benzylhydroxamate of 2, was synthesized byN-acylation of N-benzylhydroxylamine hydrochloride with 2 activated by(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent) in N,N-diiso-propylethylamine (DIEA; 3 equiv) and DMF(Scheme 8). Although it was possible to isolate an optically activehydroxamate via Sephadex LH-20 chromatography, the compound epimerizedon recrystallization. The magnitude of optical rotation decreased from[α]_(D)=−16.4° to essentially zero. Because of the spontaneousepimerization, no attempt was made to evaluate the biological propertiesof optically active materials. The racemic product was utilized in allof the studies.

Example 4 Synthesis of compounds 29, 29a, 33, 35 and 38

Compound 29 was synthesized by condensation of 2,5-dihydroxybenzonitrile

with D-cysteine as described in Scheme 1. This aryl nitrile, in turn,was made by heating 2,5-dihydroxybenzaldehyde with nitroethane in sodiumand acetic acid.

By analogy to the synthesis of 5′-hydroxydesazadesmethylDFT 29,(S)-4,5-dihydro-2-(2,5-dihydroxyphenyl)-4-methyl-4-thiazolecarboxylicacid (5′-hydroxydesazaDFT, 33) is generated from the cyclocondensationof (S)-α-methyl cysteine with 2,5-dihydroxybenzonitrile in bufferedaqueous CH₃OH. Isomeric dihydroxy desazadesmethylDFT 35 is synthesizedfrom the requisite aryl nitrites and D-cysteine in methanolic phosphatebuffer (pH 6). Dihydroxy desazaDFT 38 is prepared by treatment of(S)-α-methyl cysteine with the same set of trihydroxybenzonitriles.Heating 2,4,5-trihydroxybenzaldehyde with nitroethane in sodium acetateand acetic acid provides the corresponding trihydroxybenzonitrile,aromatic precursors to compounds 35 and 38.

(S)-4,5-Dihydro-2-(5-fluoro-2-hydroxyphenyl)-4-thiazolecarboxylic acid(5′-fluorodesazadesmethylDFT, 29a) is prepared from the cyclization ofD-cysteine onto 5-fluoro-2-hydroxybenzonitrile in slightly acidicbuffer. The aromatic precursor could be made by direct cyanation ofp-fluorophenol using methyl thiocyanate, aluminum chloride, and borontrichloride in ethylene dichloride followed by heating in aqueous base(Adachi et al., Syn. Commun. 20:71-84 (1990)).

Example 5 Synthesis of Compounds 40-43

The synthesis of bis-DFT compounds 40 and 41 is dependent on therelative acidity of the C-2 proton of 1,3-dimethoxybenzene (51), whichhas been metalated (n-BuLi/THF) then alkylated in the 2-position with1-bromopropane (Brown et al., J. Med. Chem. 32: 807-826 (1989) and1,4-diiodobutane (Tanaka et al., Chem. Lett.: 1905-1908 (1989)) in thelatter case joining two aromatic rings. Thus, deprotonation andregiospecific alkylation of 51 with 1,9-dichlorononane (52a) or1,11-dibromoundecane (52b), respectively, affords tetramethoxy compounds53a,b (Scheme 3). The four methyl protecting groups are removed withBBr₃/CH₂Cl₂, yielding tetraphenols 54a,b. Vilsmeier-Haack formylationortho to a phenol of each ring (POCl₃/DMF/CH₃CN) gives dialdehydes 55a,bwhich are directly converted to dinitriles 56a,b with nitroethane/NaOAcin HOAc (Karmarkar et al., Synthesis: 510-512 (1985). We have recentlyformylated 2-methylresorcinol under these conditions to yield2,4-dihydroxy-3-methylbenzaldehyde, which was carried through to(S)-4,5-dihydro-2-(2,4-dihydroxy-3-methylphenyl)-4-thiazolecarboxylicacid. To complete the synthesis of hexadentate chelators 40 and 41, bisnitriles 56a,b are reacted by D-cysteine (>2 equiv) in pH 6 buffer.

The analogous ether-containing ligands 42 and 43 are synthesizedstarting with 1,3-bix(benzyloxy)benzene (57) (Haraldsson et al.,Tetrahedron 53: 215-224 (1997)); which is methylated then alkylated inthe 2-position with 0.5 equivalent of 4-chlorobutyl ether (58a) ortetra(ethylene glycol) di-p-tosylate (58b), respectively (Scheme 4). Thephenols are protected by benzyl groups instead of methyls sincetreatment with BBr₃ would cleave the ether-containing tethers along withthe methyl ethers. The resulting symmetrical compounds 59a,b arecatalytically deprotected under mild conditions (1 atm, Pd/C, CH₃OH),affording tetraphenols 60a,b. Completion of the synthesis of 42 and 43is carried out as in Scheme 3.

Example 6 Synthesis of Compounds 44-50

The preparation of bis DFT compounds 44 and 45 depends on the ability toacylate resorcinol (61) at C-4 (Scheme 5). Specifically, heating sebacicacid (62a) or undecanedioic acid (62b) with 61 intrifluoromethanesulfonic acid (Koch et al., J. Org. Chem. 59:1216-1218(1994)) gives diketones 63a,b. The carbonyl groups are then changed intomethylenes with hydrogen (PdC/AcOH) (Horning et al., J. Am. Chem. Soc.71:1036-1037 (1949)) providing tetraphenols 64a,b. The Vilsmeier-Haackreaction leads to dialdehydes 65a,b such that the connector is in the5-position of the rings. After conversion to the dinitriles 66a,b asabove, cyclocondensation with D-cysteine (>2 equiv) under weakly acidicconditions generates 44 and 45, respectively.

The oxa substituted tethers for chelators 46 and 47 are made in astepwise manner (Scheme 6). 3-(2,4-Dihydroxyphenyl)propionic acid (67)is transformed into the tribenzyl derivative 68; reduction of the esterwith LiAlH₄ in THF provides carbinol 69, in which the phenols remainbenzyl protected (Amsberry et al., J. Org. Chem. 55: 5867-5877 (1990)).Two equivalents of primary alcohol 69 are linked by treatment of itsalkoxide with ethylene diiodide (70a) or 1,3-diiodopropane (70b).Adducts 71a,b are then catalytically debenzylated to afford diethers72a,b. Completion of the synthesis of compounds 46 and 47 isaccomplished using the

methodology of Scheme 5.

Coupling of diacid 47 with N-methylhydroxylamine (2 equiv) using BOPreagent (excess DIEA, DMF) (Bergeron et al., J. Med. Chem. 37:1411-1417(1994)) provides bis-hydroxamate 48. N-Acylating dihydroxylamine 73 withthe same dicarboxylic acid (1:1) and coupling reagents under highdilution conditions leads to macrocyclic chelator 49. Analogous reactionof the dihydroxylamine K′ (Bergeron et al., J. Med. Chem. 42: 2881-2886(1999)) with 2 equivalents of 4′-hydroxydesazadesmethylDFT (25)(Bergeron et al., J. Med. Chem. 42; 95-108 (1999)) generates hexadentatechelator 50 (Scheme 7).

Example 7 Synthesis of Compound 25

Compound 25 was generated by N-acylation of N-aklyhydroxylamines (K)with (S)-desmethylDFT (2) (Scheme 9) (Bergeron et al., J. Med. Chem.37:1411-1417 (1994); Bergeron et al., J. Med. Chem. 42:2881-2886(1999)). Hexacoordinate compound 25,(S,S)-N¹,N⁸-bis[4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-thiazoyl]-N¹,N⁸-dihydroxy-3,6-dioxa-1,8-octanediamine,resulted from N-acylation of N dihydroxy-3,6-dioxa-1,8-octanediamine ateach terminus with acid 2 using BOP.

Example 8 Compounds Useful in the Invention

A. Preparation of a compound of formula (I) where A is CH; each of R₁,R₂, R₃, R₄, R₅, R₆ and R₁₁ is H; n is 0; p is 0; and C(O)R is COOH.

2,4-Dihydroxybenzonitrile was prepared according to the method of Marcusin Ber. Dtsch. Chem. Ges. 1981, 24, 3651, as follows:

A mixture of 2,4-dihydroxybenzaldehyde (5.0 g, 36.2 mmol), sodiumacetate (5.94 g, 72.4 mmol), nitroethane (5.44 g, 72.4 mmol) and glacialacetic acid (10 ml) was refluxed for 6 hours. After cooling, the mixturewas poured onto ice (100 g) and extracted with ethyl acetate (4×50 ml).The combined organic layers were washed with saturated NaHCO₃ until thepH of the aqueous layer remained at 8, dried (Na₂SO₄), and the solventremoved in vacuo. Flash chromotography (SiO₂, cyclohexane:ethylacetate—1:1) afforded 2,4-dihydroxybenzonitrile (2.87 g, 59%) as a paleyellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 6.33 (d, 1H, J=8.6 Hz), 6.43(s, 1H), 7.37 (d, 1H, J=8.6 Hz), 10.35 (s, 1H), 10.78 (s, 1H). IR (KBr)2200 cm⁻¹.

D-cysteine hydrochloride monohydrate (6.8 g, 38.7 Phenol) was added to asolution of 2,4-dihydroxybenzonitrile (3.5 g, 25.9 mmol), prepared asdescribed above, in a mixture of degassed methanol (1105 ml) and 0.1 Mphosphate buffer, pH 5.95 (70 ml). NaHCO₃ (3.25 g, 38.7 mmol) wascarefully added and the mixture was stirred at 70° C. under Argon for 54hours. Volatile components were removed under reduced pressure and thesolution was acidified with 1 N HCl to pH 2. The resulting brownprecipitate was vacuum filtered and the solid washed with water (40 ml)and ethanol (20 ml). The crude product was dissolved in saturated NaHCO₃(700 ml) and the aqueous solution washed with ethyl acetate (2×200 ml).The aqueous layer was filtered through a fine frit and acidified with 1N HCl to pH 2. The precipitated product was vacuum filtered. The aqueouslayer was extracted with ethyl acetate (4×400 ml), the combined organicextracts were dried (Na₂SO₄) and the solvent was removed in vacuo. Theremaining solid was combined with the precipitated product and driedunder high vacuum at 40° C. for 12 hours to give4,5-dihydro-2-(2,4-dihydroxyphenyl)-thiazole-4(S)-carboxylic acid (4.08μg, 66%), mp 266-268° C. (dec) (Ind. J. Chem., Vol. 15B, Kishore et al,pages 255-257 (1977) for (L)-isomer: 261-262° C.). ¹H NMR (300 MHz,DMSO-d₆) δ 3.61 (m, 2H), 5.38 (dd, 1H, J=7.2/9.4 Hz), 6.31 (d, 1H, J=2.3Hz), 6.38 (dd, 1H, J=2.3/8.6 Hz), 7.25 (d, 1H, J=8.6 Hz), 10.25 (br s,1H), 12.60 (br s, 1H), 13.15 (br s, 1H). Anal. Calc. For C₁₀H₉NO₄S: C,50.20; H, 3.79; N, 5.85. Found: C, 50.13; H, 3.82; N, 5.85.

B. Preparation of a Compound of Formula (I) where A is N; each of R₁,R₂, R₃, R₄, R₅, R₆ and R₁₁, is H; n is 0, p is O and C(O)R is COOH.

By following the procedure of Part A of this example, but substitutingthe corresponding pyridyl aldehyde for 2,4-dihydroxybenzaldehyde, thecorresponding pyridyl compound:4,5-dihydro-2-(3′,5′-dihydroxypyrid-2′-yl)-thiazole-4(S)-carboxylicacid.

Example 9 Compound Useful in the Invention

A. Preparation of a Compound (S)-desmethyldesferrithiocin,N-methylhydroxamate.

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP) (442.3 mg, 1.0 mmol) was added to a solution of(S)-desmethyldesferithiocin [See Example 1] (224.2 mg, 1.0 mmol) andN-methylhydroxylamine hydrochloride (83.52 mg, 1.0 mmol) indimethylformamide (DMF) (8 ml) at 0° C. A solution ofdiisopropylethylamine (DIEA, 129.2 mg, 1.0 mmol) in DMF (2 ml) was addeddropwise to the above solution at 0° C. The mixture was stirred at 0° C.for 15 minutes and at room temperature overnight. Solvent was removedunder high vacuum and the residue was treated with ethyl acetate (EtOAc,30 ml). The organic phase washed with 10 ml portions of saturatedNaHCO₃, saturated NaCl, 10% citric acid and saturated NaCl, and solventwas removed by rotary evaporation. Purification of the residue on aSephadex LH-20 column, eluting with 3% EtOH/toluene, produced 120 mg of(S)-desmethyldesferrithiocin, N-methylhydroxamate (47%) as a yellowsolid: α25-41.3° (c 2.34); NMR (CDCl₃/d₆DMSO) δ 3.27 (s, 3H) 3.53 (dd,2H, J=9, 6), 5.70 (t, 1H, J=9), 7.30 (d, 2H, J=3), 8.10 (t, 1H, J=3).Anal. calculated for (C₁₀H₁₁N₂O₃S): C, 47.42, H, 4.38; N, 16.59 found:C, 47.66; H, 4.41; N, 16.45.

Example 10 Animal Models

One screen for efficacy is the normal (not iron-loaded) Sprague-Dawleyrat with a cannulated bile duct. In this model, collection of bile andurine after oral or parenteral administration of a chelator permits therapid determination of the magnitude and route(s) of excretion. Theiron-loaded Cebus apella monkey is used as a better screen, however. Todate, the behavior of iron chelators in this primate model hasbeen-quantitatively predictive of both the magnitude and routes of ironclearance after human administration.

The primary measure of activity is the efficiency of the compound, asassessed in both rodent and primate models and compared with sc DFO.Compound efficiency is a comparative evaluation of how much ironexcretion a chelator promotes relative to the theoretical amount. Forexample, the hexacoordinate chelator DFO forms 1:1 iron complexes with aformation constant of 3×10³⁰ M⁻¹ (Anderegg et al., Helv. Chim. Acta 46:1409-1422 (1963)); if the efficiency were 100%, one mmol of DFOadministered to an animal would cause one mmol of the iron complex to beexcreted. In fact, only 5% of the calculated iron is excreted when DFOis administered to humans (Kirking et al., Clin. Pharmacol. 10:775-783(1991)). In the case of the DFT analogues, the efficiency calculation isbased on the formation of a 2:1 complex with a formation constantassumed to be similar to that of the parent compound, 4×10²⁹ M⁻¹(Anderegg et al., J. Chem. Soc., Chem. Commun. 1194-1196 (1990).

The results are shown below in Table II.

TABLE II % Efficiency of Iron Clearance Compound Rat Monkey  1(S)  5.5 ±3.2 16.1 ± 8.5 (150 μmol/kg po)  2(S)  2.4 ± 0.56 (po) 4.8 ± 2.7 (150μmol/kg po)  1.8 ± 0.7 (sc) 8.0 ± 2.5 (300 μmol/kg po) 8.3 ± 2.7 (300μmol/kg sc)  2(R)  3.9 ± 1.8 0.5 ± 2 (300 μmol/kg po)  3(S)  1.4 ± 0.6(po) 12.4 ± 7.6 (300 μmol/kg po)  3(R)  4.2 ± 1.6 (po) 8.2 ± 3.2 (300μmol/kg po) 15(S) ≦0.5 16(S) ≦0.5 17(S) ≦0.5  9(S)  2.7 ± 0.5 (po) 21.5± 12 (75 μmol/kg po) 13.1 ± 4 (300 μmol/kg po) 43.3 ± 8.5 (300 μmol/kgsc) 14(S) ≦0.5  5(S)  2.4 ± 0.9 (po) 4.2 ± 1.4 (150 μmol/kg po) 5.6 ±0.9 (150 μmol/kg in H₂O sc)  5(S) 5.3 ± 1.7 (300 μmol/kg po) 4.8 ± 1.4(300 μmol/kg in H₂O po)  5(R) ≦0.5 1.7 ± 0.8 (150 μmol/kg po) 18  2.9 ±1.3 0.7 ± 0.3 (300 μmol/kg po) 19  3.7 ± 1.1 2.1 ± 0.7 (300 μmol/kg po)20 12.3 ± 3.2 ≦0.5 (75 μmol/kg po) 21  5.9 ± 3.2 3.5 ± 1.8 (150 μmol/kgpo)  6(S)  0.9 ± 0.3 (po)  0.5 ± 0.9 (sc)  7(S) ≦0.5 28(S) ≦0.5 17.7 ±3.9 (75 μmol/kg po) 13.4 ± 5.8 (150 μmol/kg po) 32  4.6 ± 2.1 (po) 20.6± 1.3 (sc) 22  3.1 ± 0.4 (po) 6.9 ± 3.0 (150 μmol/kg po)  5.3 ± 0.7 (sc)13.2 ± 7.7 (300 μmol/kg po) 11.7 ± 5.5 (150 μmol/kg sc) 23  2.8 ± 0.8(po) 3.2 ± 2.0 (225 μmol/kg po)  8.5 ± 0.4 (sc) 12.8 ± 3.4 (225 μmol/kgsc) 24   1 ± 0.1 (po) ≦0.5 (300 μmol/kg po)  1.4 ± 0.8 (sc) ≦0.5 (300μmol/kg sc) 25  4.6 ± 2.1 (po) 20.6 ± 1.3 (sc) po (oral) and sc(subcutaneous) refer to the route of administration

Example 11 Impact of Desferrithiocin Analogs on Malarial Parasites InVitro

Studies can be done on the developmental stages and morphology of themalarial parasite in vitro; the minimum effective concentration requiredfor cytotoxicity can be shown. The target structures include the nuclearenvelope, the membrane of the food vacuole, accumulation ofelectron-dense substances in the mitochondria, undigested materials, anddilation of the endoplasmic reticulum.

In order to determine the concentration at which a compound iscytostatic and cytotoxic, the parasites are exposed to the drug using atleast two different concentrations. After 48 h the chelator is removed,and the parasites are incubated for an additional 48 h. The percentparasitemia is determined at both time points (48 and 96 h). The effectsof the various compounds on malarial parasites seem quite varied. Forthe purposes of comparison, DFO can serve as the positive control. DFOinhibits malarial growth during an initial 48-h continuous exposure at10⁻⁵ M, normal growth resumes after the compound is removed. However, ata concentration of 10⁻⁴ M, DFO is cytotoxic; the plasmodia die duringthe 48-h incubation with the compound.

The compounds designated as 8 and 22 in Table I are active in this invitro assay. Others can be similarly tested.

1. A pharmaceutical composition comprising an amount of a compoundrepresented by formula (I) sufficient to reduce the population ofPlasmodium in a human infested with Plasmodium protozoans and sufferingfrom malaria, in combination with a pharmaceutically acceptableexcipient, wherein formula (I) is

where: R is OH, OR₇, or N(OH)R₈; R₁ is H, CH₃ or an available electron;R₂ is H, CH₃ or an available electron; R₃ is H, CH₃ or an availableelectron and together with either R₁ or R₂ when one is an availableelectron, forms a double bond with the R₁/R₂ carbon; R₄ is H, acyl of1-4 carbons or alkyl of 1-4 carbons; R₅ is H, OH, O-acyl of 1-4 carbons,O-alkyl of 1-4 carbons, or (CH₂)_(a)(R₁₀)_(b)(CH₂)_(a)R₁₀(CH₂)_(a)(R₁₀)_(b)X R₆ is H, OH, alkyl of 1-6 carbons, a halogen,(CH₂)_(a)(R₁₀)(CH₂)_(r)(R₁₀)Y, or is C═C—C═C—, which, together with R₁₁,when R₁₁, is an available electron, forms a fused ring system asfollows:

R₇ is alkyl of one to four carbons or optionally substituted benzyl; R₈is H, alkyl of one to four carbons, optionally substituted benzyl,

R₉ is H, alkyl of one to four carbons or optionally substituted benzyl;R₁₀ is O or CH₂, R₁₁ is H, OH, O-acyl of 1-4 carbons, O-alkyl of 1-4carbons or an available electron; A is N, CH or COH; B is S, O, N, CH₂or CH₂S; a is 2 or 3; b is 0 or 1; m is an integer from 1 to 8; n is 0or 1; p is 0, 1 or 2; r is 2 or 3;

wherein each of the substituents shown is defined above, or a compoundof formula (I) where the ring containing the B and N moieties is fullyreduced and contains no double bonds, or a pharmaceutically acceptablesalt of the compound represented by formula (I) or a stereoisomer of thecompound or mixture of stereoisomers.
 2. The composition of claim 1,wherein B is S, and n and p are
 0. 3. The composition of claim 2,wherein each of R₁, R₂ and R₃ is H or CH₃, each of R₅ and R₁₁ is H, OH,O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons, R₆ is H, OH, alkyl of1-6 carbons or a halogen, and R₇ is alkyl of 1-4 carbons.
 4. Thecomposition of claim 3, wherein R₄ is H.
 5. The composition of claim 2,wherein R is N(OH)R₈.
 6. The composition of claim 5, wherein R₈ is CH₃.7. The composition of claim 1, wherein A is CH.
 8. The composition ofclaim 7, wherein B is S, and n and p each is
 0. 9. The composition ofclaim 8, wherein each of R₁, R₂, R₃, R₄ is H or CH₃, each of R₅ and R₁₁is H, OH, O-acyl of 1-4 carbons or O-alkyl of 1-4 carbons, R₆ is H, OH,alkyl of 1-6 carbons or a halogen, and R₇ is alkyl of 1-4 carbons. 10.The composition of claim 9, wherein R₄ is H.
 11. The composition ofclaim 9, wherein each of R₁, R₂, R₃, R₅, and R₆ is H.
 12. Thecomposition of claim 9, wherein R₁₁ is OH.
 13. The composition of claim12, wherein each of R₁, R₂, R₃, R₅, and R₆ is H.
 14. The composition ofclaim 10, wherein R is N(OH)R₈.
 15. The composition of claim 14, whereinR₁₁ is OH, and each of R₁ and R₂ is H.
 16. The composition of claim 15,wherein each of R₃ and R₅ is H.
 17. The composition of claim 15, whereinR₈ is CH₃.